PQ kit
for HPLC Compliance
PQ kit
TM
TM
for HPLC Compliance
Operating Procedures—Quick Reference Introduction
This document is designed as a “Quick-Start” instruction summary to help you quickly get started on an HPLC Performance Qualification. Refer to the following Primary Documents as necessary for a more detailed discussion of the Test Protocols: Operating Instrucftions: Detailed procedures to carry out the selected Performance Qualification Test Protocols. Test Protocols: Background theory, calculations and in-depth discussion of operating principles. Summary Form: HPLC Performance Qualification Summary Form. Exceltm Template: Excel-based software to calculate, graph and summarize results.
Preparations
Step 1: Perform any required Instrument Maintenance. A Performance Qualification is a System Test, designed to qualify an HPLC that has been serviced, and is in generally good repair. Prior to starting, ensure that lamps, seals, check valves, rotor seals and other typical maintenance items have been serviced according to the Instrument manual, and/or your relevant SOP’s. Step 2: The pump Flow Rate must be verified for an accuracy of ± 5% or better, at a flow rate of 1.0 mL/min, with the PQ Test Column in place, with the Method Mobile Phase. If the pump has already been qualified at this flow rate, you may enter the actual flow value into the software from the calibration records. If not, use a dry 5 mL volumetric flask and calibrated stopwatch to confirm the flow accuracy at 1.0 mL/min. Note that this flow rate check is not meant to replace the typically more extensive Operational Qualification checks at several flow rates. Rather, it is to document the Suitability of the pump to perform the various Performance Qualification Test Protocols described in the kit. Step 3: Prepare Mobile Phase(s) 13:87 Acetonitrile:0.1% Acetic Acid (1 mL/L). One Liter is sufficient for a simple isocratic qualification. For a quaternary gradient, 2 L is recommended. Mobile phase is stable for 60 days, if sealed. Flush and equlibrate HPLC using the PQ Test Column. If a Gradient Qualification is to be performed, reserve about 500 mL of mobile phase. Spike with Gradient Visualization Solution (GVS) at the rate of 6 mL/L, according to the Operating Instructions. Use this only in the “B*” solvent reservoir. Step 4: Set up Methods and Sequence according to the detailed requirements of your HPLC and Data System, and following your company’s internal SOP’s regarding documentation. Injection Sequence 01 shows an example of a typical Sequence performing all the Test Protocols. Adapt this Sequence to your particular instrument as necessary. For example, if only a single injection per vial is used, then multiple vials must be prepared rather than a single vial. Except for the Primary Standard Wavelength Calibration with Holmium Oxide solution, all other tests are fully automated. Once the Sequence is designed, it should operate without user intervention.The various methods all utilize the same general instrument conditions. These are: Method Wavelength: 273 nm Injection Volume: 10 µL (except for volume linearity) Flow Rate: 1 mL/min Temperature: Ambient Note that column oven temperature is not qualified with the PQ Kit, since most companies maintain their own specific SOP’s addressing this component. The use of a column oven is not required for the PQ Tests. The operating temperature for the PQ Tests should be within the range of 15°C-27°C (60°F-80°F).
MICROSOLV TECHNOLOGY CORPORATION 103 Brighton Ave. • Long Branch, NJ 07740 USA 732-229-3400 • www.MicroSolvTech.com
PQ kit
TM
for HPLC Compliance
Operating Procedures—Quick Reference Page 2
Step 5: After the Test Protocols have been completed, enter the results into the provided Excel™ Template. There are extensive drop-down menus and instruction to help with data entry. Note that the first page of the spreadsheet contains both Instrument data and Test Result Summaries. Note that the data are automatically listed in this section only for the test protocols actually performed. Another summary sheet, Form 3.0, provided in Word and Adobe formats, can be copied and adapted to your laboratory, with your company’s name and other information, to provide a convenient single-page form to easily summarize the test results for each HPLC tested. This form can be incorporated into your internal SOP’s. Sequence 01:
Line No.
General HPLC Pe rformance Qualification Example Injection Sequence
Sample N ame
Method
Test Protocols: System Suitability: Dynamic Short Term Noise : HPLC Extra-Column Volume Variance :
# Inj
Requires:
Comments:
1 vial Mo bile Phase Blan k 1 vial Resolution Test Mix
1
Mobile Phase Blank
RTM
2
1 clean blank is sufficient. However, 2 or more are recommended to ensure a clean instrument.
2
Resolution Test Mixture
RTM
1
Used for System Suitability and Extra-Column Volume test.
AutosamplerInjection Precision : Flow Rate (Retention Time) Precision:
Requires:
1 vial soluti on L3 (10 vials required fo r single injection/vial)
3
Linearity Solution L3
PQ10
10
Autosampler precision and System drift
4
Mobile Phase Blank
PQ10
2
Ensures clean system prior to starting Linearity
Detector Respo nse Linearity 100% - 0.1% : Injector % Car ryover :
5
Linearity Solution L1 (0.1%)
6
Linearity Solution L2
7 8
Requires:
PQ10
3
(1%)
PQ10
3
Linearity Solution L3 (20%)
PQ10
3
Linearity Solution L4 (50%)
PQ10
3
9
Linearity Solution L5 (75%)
PQ10
3
10
Linearity Solution L6 (100%)
PQ10
3
11
Mobile Phase Blank
PQ10
3
Autosampler Injection Volume Linearity :
Requires: Note:
1 vial each Linearity Solution (3 vials each for single injection/vial) 1 fresh vial Mobile Phase for % Car ryover Begin Detector Linearity with 0.1% solution
Triplicate injections at each level
Conclude with 100% level solution, L6
Use area of peak eluting at tR of Caffeine in 1st Blank Injection for % Carryover. Examine inj 2 and 3 for instrument trends. 1 vial of L inearity Solution L3 pe r Volume Tested (3 vials each for single injection/vial) Solution L3 is within the System Linear Range for 1-50 :L. Use L2 if testin g larger volumes.
12
Linearity Solution L3*
PQ05
3
Autosampler Volume Delivery Linearity.
13
Linearity Solution L3*
PQ10
3
14
Linearity Solution L3*
PQ20
3
15
Linearity Solution L3*
PQ40
3
Methods PQ05, PQ10, PQ50.... are the same as PQ10 method, but modified for injection volume only. Select any 5 injection volumes over the range of 1 - 50 : L if solution L3 is used, to remain within detector linear range.
16
Linearity Solution L3*
PQ50
3
MICROSOLV TECHNOLOGY CORPORATION 103 Brighton Ave. • Long Branch, NJ 07740 USA 732-229-3400 • www.MicroSolvTech.com
PQ kit
TM
for HPLC Compliance Sequence 01:
Line No. 17
Operating Procedures—Quick Reference Page 3
General HPLC Pe rformance Qualification Example Injection Sequence
Sample N ame Mobile Phase Blank
Method
# Inj
PQ10
2
Gradient Delivery Accuracy and Gradient Dwell Volume:
Requires:
Comments: Cleans system prior to start of Gradient Tests
1 vial of GVS spiked B* mobile phase for Dwell Volume (use the A/B* combination) 1 vial Blank Mobile Phase (unspiked) for others (ternary, quaternary).
18
GVS Spiked Mobile Phase
GRDAB
1
Determine Gradient Dwell Volume and Gradient Delivery Accuracy for solvent combination A/B*, where B* is spiked with GVS (for binary gradient systems).
19
Mobile Phase Blank
GRDCB
1
Determine Gradient Delivery Accuracy for solvent combination C/B* (for ternary gradient systems)
20
Mobile Phase Blank
GRDDB
1
Determine Gradient Delivery Accuracy for solvent combination D/B* (for quaternary gradient systems)
Wavelength Accuracy - Primary Standard Method: (NIST-Traceable Holmium Oxide) For Diode Array Detectors Step 1: Setup Wavelength Accuracy HPLC Acquisition method using 0 Flow and No Injection. Monitor at 241 nm.
Step 2: Manually fill flow cell with Purified Water, by pulling it from the column outlet tubing using the syringe provided. Step 3: Start the Wavelength Accuracy HPLC Acquisition method with Purified Water in the flow cell. The detector should reference/balance against water at the beginning of the injection. Step 4: Manually fill flow cell with Wavelength Calibration Solution, by pulling it from the outlet tubing using the syringe provided. Step 5: After cell is filled, disconnect syringe; let signal stabilize. Collect the spectra of the Wavelength Calibration Solution in the stable plateau region of the Holmium Oxide signal. For Variable Wavelength Detectors: Step 1: Manually fill flow cell with Wavelength Calibration Solution, by pulling it from the column outlet using the syringe provided. Step 2: Zero the detector at 590 n, a region of no absorbance (see Holmium Oxide spectrum). Step 3: Select up to 5 calibration wavelengths, in the spectral region of interest (UV, Vis, or Both). Step 4: Document absorbance readings in 1 nm increments several nm above and below the bands of interest. Wavelength Accuracy - Secondary Standard Method: (NIST-Traceable Caffeine) Step 1: Acquire spectrum of Caffeine in Linearity Solution L3, using Diode Array, or Variable Wavelength Detector at multiple wavelengths above and below 273 nm. Set up multiple methods, all identical except for wavelength. Certified Wavelength of Caffeine under the Method Mobile phase is 273 nm ± 1 nm.
MICROSOLV TECHNOLOGY CORPORATION 103 Brighton Ave. • Long Branch, NJ 07740 USA 732-229-3400 • www.MicroSolvTech.com
